Method of evaluating metabolism of the eye

ABSTRACT

A method and apparatus for measuring the retinal auto-fluorescence of a subject retina includes an excitation light source for providing an excitation light at a wavelength of at least 450 nm and an image capture device for recording an ocular auto-fluorescence signal generated in response to the excitation light. The image capture device includes a filter for reducing background non-signal wavelengths from the ocular auto-fluorescence signal and an image intensifier for increasing the ocular auto-fluorescence signal strength. The method and apparatus may further include a processor that analyzes the ocular auto-fluorescence signal to determine a contrast change or pattern to thereby detect retinal disease or damage. The processor may compare the images with control images, past images of the same eye or other diagnostic modalities such as fundus photography, angiography, or visual field testing to detect the retinal disease or damage.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA074120 andEY009441, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

TECHNICAL FIELD

The disclosed device generally relates to measuring characteristicswithin the retina. In particular, the device embodies a non-invasive,single image method and apparatus for measuring metabolic activitywithin the retina and optic nerve.

BACKGROUND

FIG. 1 illustrates an exemplary eye 10 including a cornea 20 and a lens22 to focus and direct light onto a retina 30, which is the lightdetection and neural processing component of the eye 10. The retina 30extends from the optic nerve 24, which is composed of retinal nervefibers, near the posterior pole 26 of the eye 10 to the or a serrata 28extremitfy near the anterior segment 32 of the eye 10. The retina 30contains two types of photoreceptor cells, rods and cones, whichgenerate electrical signals in response to light.

Failure of any retinal component may result in blindness. For example,total or partial blindness may be caused by a reduction in blood supplyto the retina, which in turn, may be the result of diabetic retinopathyor ischemic events such as retinal vein occlusion. Research has shownthat other causes of blindness such as cytomegalovirus retinitis,glaucoma, Leber's optic neuropathy, retina detachment, age-relatedmacular degeneration, retinitis pigmentosa, or light induced blindnessare commonly associated with the apoptotic, or programmed death ofretina cells.

Apoptosis generally involves the activation of one or more apoptoticsignaling pathways by intrinsic or extrinsic stimuli causing theselective degeneration of neurons. The onset of apoptosis has beenlinked to mitochondrial dysfunction (which is indicative of a change incellular metabolic activity) characterized by the loss of mitochondrialintegrity leading to the release of apoptotic mediators and theactivation of enzymes and other pathways leading to cell death. Thesechanges in mitochondrial integrity result in a gain or a loss of pro-and anti-apoptotic signals and have been linked to the retina disordersthat result in 95% of the instances of irreversible blindness. Earlydetection of mitochondrial dysfunction can allow for diagnosis,treatment, and monitoring of these disorders.

Current diagnostic techniques used in routine eye examinations typicallyemploy ophthalmoscopes to visually inspect the retina and tonometers toevaluate intraocular pressures. While ophthalmoscopes can be used todiagnose retinal degeneration, they are only effective after substantialdamage has already occurred and do not provide any indication ofmitochondrial activity. Tonometers indent the eye in order to determinechanges in intraocular pressure that may result in glaucoma, retinalganglion cell death, or ischemia. However, the correlation betweenintraocular pressure and disease is not robust, as evidenced by patientsdeveloping glaucomatous degeneration with low pressures and patientswith high pressure remaining disease free. Furthermore, these oldermethods cannot be correctly interpreted in the presence of biomechanicalartifacts such as abnormal corneal thickness due to, for example,natural variations, disease, myopia, or refractive corneal surgery.

U.S. Pat. No. 4,569,354 entitled “Method and Apparatus for Measuring theNatural Retinal Fluorescence” discloses a method and apparatus fordetermining oxygenation of the retina by measuring the fluorescence offlavoprotein in the retina. According to this patent, a spot ofexcitation light of a wavelength of about 450 nanometers (nm) is scannedacross the retina, in response to which retina auto-fluorescence at awavelength of about 520 nm is detected. In particular, retinal emissionlight is detected at two wavelengths of about 520 nm and 540 nm to allowfor compensation with respect to absorption and transmission variablesin the eye. To compensate for fluorescence of the lens of the eye, thecenter of the pupil is imaged onto scanning mirrors so that the scanningbeam of excitation light pivots at the center of the eye lens. Becausethis method and apparatus scans a small area of the retina (i.e. a verylimited number of pixels) at a time, the strength of the measured signalis extremely low, resulting in a measured signal having a lowsignal-to-noise (S/N) ratio and little, if any, accuracy. Further, thesmall scan area necessitates an extended procedure time to completelyscan the retina, which further increases potential for error caused byeye movement due to natural instability of extraocular muscle tone,blood pulsation and light contamination. Because of the inherentinaccuracies of this method and device, it is unable to operate as anaccurate diagnosis and monitoring system.

Accordingly, a device and method for measuring the metabolism of the eyeis needed to address the shortcomings of the known diagnostic tools andmethods discussed above. Specifically, a device and method fornon-invasively measuring the metabolic activity of cells that increasesthe diagnostic accuracy and speed in detecting retinal disorders isneeded.

SUMMARY

The method and apparatus disclosed herein provides a rapid andnon-invasive clinical and experimental tool to measure directly thevitality of a retinal cell based on the auto-fluorescence of excitedflavoprotein (FP) within the retinal mitochondria. The disclosed methodand apparatus for measuring the retinal auto-fluorescence of a subjectretina includes an excitation light source for providing an excitationlight at a wavelength of approximately 450 nm and an image capturedevice for recording an ocular auto-fluorescence signal generated inresponse to the excitation light. The image capture device includes afilter for filtering out undesired wavelengths from the ocularauto-fluorescence signal and includes an image intensifier forincreasing the ocular auto-fluorescence signal strength. The method andapparatus may further include a processor that analyzes the ocularauto-fluorescence signal to determine a contrast change or pattern andcan compare serial readings taken at different times or dates. Salientobjectives addressed by the device and method disclosed below include:fast procedure time, high accuracy, a direct correlation between retinalmetabolic activity and the existence of a retinal disorder, andincreased signal-to-noise ratio (S/N).

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the disclosed device, referenceshould be made to the following detailed description and accompanyingdrawings wherein:

FIG. 1 illustrates a cross-sectional view of the components of anexemplary eye;

FIG. 2 illustrates a flowchart embodying the operation of an exemplaryretinal testing and evaluation apparatus;

FIG. 3 illustrates a flowchart embodying the operation of an exemplarycalibration routine; and

FIG. 4 illustrates the functional arrangement of a plurality ofcomponents included with an exemplary evaluation apparatus.

DETAILED DESCRIPTION

Cellular auto-fluorescence may be observed by measuring the emissions ofendogenous flavoproteins (FPs) and NAD(P)H molecules. Previouslaboratory studies and experiments have shown that cellular metabolicactivity is related to the auto-fluorescence of both FP and NAD(P)Hmolecules. However, NAD(P)H excites and auto-fluoresces in the nearultra-violet range, which promotes cataracts and retinal damage and is,therefore, not suitable for use on living subjects. Therefore, FPauto-fluorescence is being evaluated using, for example, a brief blueexcitation light that can be transmitted via the optical structures ofthe eye without risk of retinal damage. Previous studies have indicatedthat elevated levels of apoptotic activity correlates with reducedmetabolic activity that increases FP auto-fluorescent intensity andreduced metabolic activity. Thus, a method and apparatus that measuresthe fluorescence of FP is useful in evaluating retinal metabolicactivity in order to aid in the early detection and/or the prevention ofblinding disorders.

FIG. 2 illustrates a flowchart embodying one possible implementation ofan exemplary retinal evaluation method 40. At a block 42, a calibrationroutine 60 (shown in more detail in FIG. 3), can be initiated eithermanually or upon the occurrence of some predefined condition. Forexample, the condition for implementing the calibration routine 60 maybe based on the non-specific autofluorescence emitted from the eye, adatabase of autofluorescence images of the same eye examined previously,a database of control eyes imaged previously, a number of cycles oroperations that the equipment has performed, the duration of time theequipment has been operating, the calculated mean time before failure(MTBF) of critical components, or any other identified criteria.

At a block 44, after the calibration routine 60 has been executed,skipped or otherwise completed, the subject to be evaluated can bepositioned or aligned relative to the evaluation equipment. It will beunderstood that the alignment procedure may be accomplished in a varietyof ways, such as employing a physical guide used on a desktopophthalmoscope, fundus camera, or slit-lamp (not shown) to align thesubject's head and retina with the equipment, a slit-lamp apparatus, ora retinal fundus camera apparatus. Further, the alignment procedure maybe implemented using software by selecting the evaluation area ofinterest from a presented digital or graphical image. By selecting thearea of interest, such as a retinal landmark like the optic disc orvascular patterns, the software may instruct a virtual camera apertureto focus or shift to the identified area of interest or may instructphysical elements within the system to shift and position into thedesired or identified location.

At a block 46, a plurality of microscope objective lenses may beadjusted to a desired focal length. These adjustments may be mademechanically using, for example, a high precision rack and pinionarrangement to linearly shift the objective lens along the same linedefined by the path of light generated by an excitation means, such as alight source. Further, it will be understood that the objective lens maybe automatically positioned using a servo or positioning motor system toshift the lens to a predetermined position relative to the physicalguide and the subject discussed in conjunction with the block 46. Inaddition, a range finder or pattern focusing technique (in which apattern is projected into the optical path and an automatic focusingroutine causes the pattern to become the system focal point) could beemployed to allow the correct focal length to be automaticallydetermined.

At a block 48, an excitation means, such as a light source, can betriggered or pulsed to stimulate the flavoproteins (FPs) associated withretinal mitochondria. Excitation may be initiated over a wide range ofwavelengths using, for example, a He—Cd or argon-ion laser, and anincandescent or mercury lamp such as an ATTOARC™ variable intensityilluminator. However it is desirable to reduce potential signal noise bylimiting the excitation spectrum to a range consistent with theexcitation spectrum of FP, approximately 460 nm. To this end, anexcitation filter such as an OMEGA OPTICAL® Model No. XF1012 (455DF70)excitation filter having a filter range of approximately 420-490 nm, maybe used. The filtered excitation means stimulates the FPauto-fluorescence without stimulating additional molecules and therebygenerating unwanted auto-fluorescence that could act as noise to degradethe overall accuracy of the evaluation technique. It will be understoodthat the excitation spectrum may be further limited by reducing theambient light adjacent to the retina 30, which may be accomplished byreducing the testing room lighting, by fitting the subject with goggles,or any other similar method.

At a block 50, a single image representing the excited FPauto-fluorescence within the retina can be captured. For example, ahigh-speed charged coupled device (CCD) camera such as a PRINCETONINSTRUMENTS® PI-MAX ICCD model 512-Gen III camera can be employed torecord an image of the auto-fluorescence of the retina. It will beunderstood that the field of view (FOV) of the CCD camera, with orwithout the magnification of the objective lens of the block 46, shouldbe established to allow imaging of the retina (or any desired portionthereof) in a single picture.

One exemplary method of choosing an appropriate FOV to be imagedincludes identifying retinal landmarks such as the optic disc orvascular patterns to use as aiming points and then adjusting the FOV toencompass the entire area of interest. Using an appropriate objectivelens or lenses, the FP auto-fluorescence may be directed onto a CCDcamera. After a set integration time, typically less than one second,the shutter is closed and the image is then downloaded to a computer.The captured digital images can be scanned visually or electronically tofind areas of increased brightness, which may be diagnosed as apoptoticregions.

The CCD camera may further be augmented with a photon intensifier suchas the above-identified PRINCETON INSTRUMENTS® image intensifier modelGen-III HQ that includes a photocathode for converting the image into anelectrical signal. The electrical signal is multiplied and acceleratedinto a phosphor screen to produce an amplified image that may then becaptured, stored and analyzed.

At a block 52, the captured amplified image can be analyzed, forexample, by a program stored in a computer memory and executed on aprocessor, to evaluate the metabolic activity within the retina asindicated by the FP signal auto fluorescence. The captured image can bevisually analyzed by an observer, trained or otherwise, to determine thepresence or absence of patterns, changes or other aberrations ofinterest. However, as described above, it may also be desirable toautomate the analysis procedure using the processor to execute imageprocessing software. A software analysis program may analyze each pixelor element of the image to individually, and in conjunction with thesurrounding pixels, determine local changes in contrast, rates of changein contrast and the existence of patterns. For example, the intensity ofa single pixel or a group of pixels may be measured and compared toanother adjacent pixel or group of pixels to determine the presence oflocal changes, patterns, or rates of change etc. which, in turn, canindicate a change in the health and function of the retina 30. Thesoftware analysis program may also correlate the captured metabolicimage patterns with photography, angiograms, or visual fieldscorresponding to the same retinal regions. In addition, the program mayanalyze historical or other stored digital images and compare them torecent or time elapsed images.

FIG. 3 illustrates the calibration routine 60 that may be implemented atany point during the retinal evaluation method 40. At a block 62, aphysical or software controlled calibration procedure can be performeddepending upon a maintenance criterion or other selection mechanism. Ata block 64, the optics and objective lens can be physically aligned byadjusting the positioning rack and pinion and/or by offsetting orshifting the initial position of a positioning servo or stepper motor.Further, the objective lenses may be rotationally shifted relative toeach other to correct for any misalignment that can cause imagedistortion, blurriness, etc.

At a block 66, the output strength and alignment of the excitation lightsource can be evaluated. The excitation light source may include, forexample, an internal photodiode that monitors the light intensity andthat operates to correct any detected variations in the output strength.Further, the photodiode may simply provide a maintenance signal toindicate when the lamp needs to be replaced. At a block 68, testinghardware, such as a guide for the subject, stand height, chin rest,forehead rest, or other testing hardware can be adjusted or aligned.

At a block 70, software or electronic calibration may be performed byexecuting diagnostic routines native to the camera and/or theintensifier. These routines may, for example, compare the stored powerlevels to detected power levels generated by the CCD camera array inresponse to a known input. At a block 72, the ambient light presentaround the retinal evaluation equipment and especially the CCD cameramay be measured. One possible manner of determining the ambient lightmay be to capture and evaluate a known image exposed to known lightingconditions with values stored within the CCD camera or another connectedprocessor, or by using a light meter to detect background ambient lightlevels at the CCD camera. The difference, if any, between the stored andthe evaluated values may then be used to offset the light and/or powerlevels of the CCD camera. At a block 74, the calibration process 60 maybe repeated based on the calculated results or other calibrationcriteria such as, for example, minimum determined CCD intensity and/orexcitation light source intensity. If the calibration procedure is notrepeated, the method may return to the retinal evaluation routine 40 asindicated.

It will be understood that the exemplary retinal evaluation method 40described above provides a non-invasive evaluation method that isclinically and experimentally useful because, among other things, themethodology is inexpensive, quick, and painless while requiring aminimum of patient effort. As discussed above, the endogenousfluorochrome flavoprotein (FP) provides an indication of the retinalmetabolic activity within retinal cells and can be monitored in areliable, non-invasive fashion.

Preliminary studies of the exemplary evaluation method 40 illustrated inFIGS. 1 and 2 included performing in vitro studies on isolated retinalcells and ex vivo studies on two isolated human retinas. The in vitroexperiments compared the auto-fluorescence excitation spectra ofpurified FP component and an unlabeled human leukocytes using a 530 nmemission wavelength. The excitation properties can be evaluated using amicrofluorometry apparatus to perform excitation spectroscopy associatedwith the 530 nm excitation emission. The in vitro experiments confirmedthat the emissions detected at the 530 nm, based on the correlationbetween the two samples, are likely to have originated with theauto-fluorescence of the flavoproteins (FPs) of interest.

The ex vivo experiments were performed on human retinal tissue having ahigh content of retinal pigment epithial (RPE) cells including oxidized,fluorescent melanin and dark granules. The physical experiment employedthe OMEGA OPTICAL® Model No. XF1012 (455DF70) excitation filter inconjunction with a 495 nm long-pass dichroic reflector. It will beunderstood that filtered excitation means may be directed and deliveredto the subject/patient using a fiber optic harness and system. Emissionspectroscopy of the excited retinal tissue showed a peak at 530 nm,which has been identified as matching the known FP auto-fluorescence.These emission results were confirmed by examining the retinal emissionspectra in the presence of a metabolic inhibitor, which caused anincrease in FP auto-fluorescence, and in the presence of cyanide, whichcaused a reduction in FP auto-fluorescence. The results of theseexperiments confirmed that emission intensity of FPs relate inverselywith the level of mitochondrial activity (e.g., an increase in metabolicactivity results in a decrease in FP auto-fluorescence).

FIG. 4 illustrates an exemplary apparatus for performing retinalevaluations generally indicated by the numeral 80. Generally, theevaluation apparatus 80 includes a still camera, a charged coupleddevice (CCD) camera 82, and an excitation light source 84 arranged tocapture a single image representing the FP auto-fluorescence presentwithin the subject retina 30. If a CCD camera 82 is used, it may be, forexample, a cooled CCD camera that may include a Peltier cooler to reducethe temperature of the detector and thereby decrease thermally generatedelectronic noise or dark current noise. As previously discussed, theretina 30 can be stimulated by the excitation light source 84 and theresulting FP auto-fluorescence can be recorded by the still camera orthe CCD camera 82. It will be understood that the CCD camera 82 can beselected to have FOV optimized to capture the single image or in thecase of a still camera, photographic image. Upon analysis, the imagecaptured by the CCD camera 82 allows a direct and non-invasive procedurefor determining the metabolic activity or health of the subject retina30.

In operation, the excitation light source 84, which may be a mercurylamp such as an ATTOARC™ mercury lamp having a bright mercury line near440 nm, or a laser of similar wave length, cooperates with a focusinglens 86 to direct the emitted excitation light 84 a to an excitationfilter 88. The excitation filter 88 may be, for example, the OMEGAOPTICAL® excitation filter described above and may be selected toprevent light of wavelengths beyond the range of approximately 400-500nm from being transmitted to the subject retina 30. The filtered light88 a may then be directed to a dichroic reflector 90, such as the 495 nmlong-pass dichroic reflector discussed above, for redirection towardsthe subject retina 30.

The redirected filtered light 88 b may then pass through an optics stage92 which may include a microscope objective 94 and a contact lens 96 ora fundus or slit-lamp camera apparatus. The microscope objective 94 andthe contact lens 96 may act to focus, align and magnify the redirectedfiltered light 88 a onto a desired area of the subject retina 30. Itwill be understood that under some test conditions, an applanation meanssuch as a flat, optically clear lens or plane may be used to flatten ordeform the cornea 20 to a desired shape to thereby allow better or moreaccurate imaging. Alternatively, an appropriate contact lens for fundusviewing may be employed.

The focused redirected light 88 c illuminates the retina thereby causingauto-fluorescence of the associated flavoproteins (FPs). The generatedFP auto-fluorescence 82 a may be directed away from the subject retina30 and through the components of the optics stage 92, and the dichroicreflector 90 to an emission filter 98 such as, for example, an OMEGAOPTICAL® Model No. XF3003 (520DF40). The emission filter 98 may beselected to prevent wavelengths that do not correspond to FPauto-fluorescence wavelength, (e.g., wavelengths of or around 530 nm)from passing through its structure. The filtered FP auto-fluorescence 82b may then pass through a focusing lens 100 which focuses FPauto-fluorescence 82 c on the still camera or CCD camera 82. At thispoint the filtered FP auto-fluorescence 82 b may be displayed on a videodisplay unit 104 such as a LCD or cathode ray tube for visualevaluation, or may be communicated to a personal computer 106 foranalysis, storage or other desired image processing.

The CCD camera 82 may further include and cooperate with an imageintensifier 102 to magnify the brightness of the focused FPauto-fluorescence 82 c to facilitate analysis of the captured image. Theimage intensifier 102 will likely be selected such that the gain, whichis the ratio between the signal captured by the detector of the CCDcamera 82 and the corresponding output signal, represents an increase of100 to 1000 times the original image intensity. The image can beacquired, for example, by using a high-speed PRINCETON ST-133 interfaceand a STANFORD RESEARCH SYSTEMS® DG-535 delay gate generator with speedsranging from 5 nsec to several minutes. The delay gate generatorcooperates with the CCD camera 82 and the image intensifier 102 tosynchronize and control the operation of these components. It will beunderstood that this captured image represents only the focused FPauto-fluorescence 82 c in an intensified form, the unwantedauto-fluorescence information or noise having been minimized by theoperation of the excitation filter 86 and the emission filter 98. Inthis manner, the resulting single image captured by CCD camera 82 has ahigh S/N ratio and provides a clear and detailed image representing theFP auto-fluorescence 82 a-82 c.

The components of the retinal evaluation apparatus 80 described hereinmay be used in a stand alone fashion, wherein alignment is accomplishedvia manual clamping and securing of the individual components. However,the imaging, excitation and optical components of the retinal evaluationapparatus 80 may be integrated into any known desktop or handheldophthalmoscope, slit-lamp, or fundus camera, to allow easy upgrade tothe testing equipment described herein. Specifically, the CCD camera 82,the excitation light source 84, the optics stage 92, and the associatedcomponents may each be equipped with an adaptor (not shown) designed toallow each of the individual components of the retinal evaluationapparatus 80 to be mated with the ophthalmoscopes and other devicesdiscussed above. In this case, the standard ophthalmoscope, fundus, orslit-lamp light may be replaced with the excitation means 84 affixed tothe ophthalmoscope frame using a bracket or adaptor and the light outputby the excitation means 84 maybe filtered to produce the desiredexcitation light 84 a. An image detection device may be attached to theframes of the devices and aligned opposite the retina 30 to detect asingle image representing the FP auto-fluorescence generated in responseto the excitation light 84 a. In this manner, existing devices can beretrofitted to allow known diagnostic equipment to be used to excite andevaluate retinal auto-fluorescence.

Although certain retinal evaluation systems and methods have beendescribed herein in accordance with the teachings of the presentdisclosure, the scope and coverage of this patent is not limitedthereto. On the contrary, this patent is intended to cover allembodiments of the teachings of the disclosure that fairly fall withinthe scope of the permissible equivalents.

1. A device for measuring apoptotic activity of an eye, said devicecomprising: an excitation light source to provide an excitation lightthat maximizes the excitation of flavoprotein auto-fluorescence in aretina and minimizes the excitation of non-flavoproteinauto-fluorescence in the retina and image capture means for recording asingle image representative of a retinal fluorescence signal generatedimmediately in response to the excitation light to minimize inaccuraciesintroduced by eye movements and rapid physiological changes, said imagecapture means including a filter to maximize the passage of flavoproteinauto-fluorescence in the retinal fluorescence signal and imageintensifier means for providing a focused amplified image showingevidence of apoptotic activity in the eye and increase the retinalfluorescence signal strength.
 2. The device of claim 1, wherein saidexcitation light source comprises a mercury lamp.
 3. The device of claim2, wherein said excitation light source comprises an excitation filterhaving a filter range corresponding to excitation of flavoproteinauto-fluorescence.
 4. The device of claim 1, wherein said excitationlight source comprises a laser.
 5. The device of claim 1, wherein saidexcitation light source is aligned with the retina using a dichroicreflector.
 6. The device of claim 1, wherein said excitation lightsource is aligned with the retina using a fiber optic system.
 7. Thedevice of claim 1, wherein said image capture means comprises a chargecoupled device.
 8. The device of claim 1, wherein said image capturemeans comprises a still camera.
 9. The device of claim 1, wherein saidimage capture means comprises a charge coupled device camera.
 10. Thedevice of claim 1, wherein said image intensifier means includes a gainfactor of at least
 100. 11. The device of claim 1, wherein said imagecapture means has a field of view sized to capture a single image of theretinal fluorescence signal generated by the retina.
 12. The device ofclaim 1, further comprising a processor programmed to analyze theretinal fluorescence signal with respect to a second stored retinalfluorescence signal.
 13. The device of claim 1, further comprising aprocessor programmed to analyze the retinal fluorescence signal todetermine a contrast change.
 14. The device of claim 13, wherein saidprocessor is programmed to analyze the retinal fluorescence signal todetermine a local contrast change.
 15. The device of claim 13, whereinsaid processor is programmed to analyze the retinal fluorescence signalto determine a rate of contrast change.
 16. A method of noninvasivelymeasuring apoptotic activity, the method comprising: providing anexcitation light generated by the excitation light source to induceretinal auto-fluorescence in a subject retina, wherein the excitationlight maximizes the excitation of flavoprotein auto-fluorescence andminimizes the excitation of non-flavoprotein auto-fluorescence;capturing a single image representing the induced retinalauto-fluorescence immediately, to minimize inaccuracies introduced byeye movements and rapid physiological changes, intensifying saidimmediately captured single image to increase the signal strength of theretinal autofluorescence; and analyzing said immediately captured singleimage to determine apoptotic activity.
 17. The method of claim 16,including aligning a still camera.
 18. The method of claim 16, includingaligning an image intensifier.
 19. The method of claim 16, includinggenerating the excitation light at an excitation wavelength of about 460nm.
 20. The method of claim 16, further including reducing the amount ofambient light presented to the subject retina.
 21. The method of claim16, further including filtering the induced retinal autofluorescence tomaximize the passage of flavoprotein auto-fluorescence and attenuatenon-flavoprotein auto-fluorescence.
 22. The method of claim 16, whereincapturing a single image includes capturing an image representative ofthe auto-fluorescence specific to flavoproteins.
 23. The method of claim16, further including analyzing the single image and comparing thesingle image with a second stored single image.
 24. The method of claim16, wherein analyzing the single image further includes determining alocal contrast change.
 25. The method of claim 16, wherein said step ofanalyzing the single image includes determining a rate of contrastchange.
 26. The method of claim 16, further including aligning at leastone objective lens between an image detection device and the subjectretina.
 27. A method of upgrading a standard imaging device tonon-invasively measure apoptotic activity of a retina, the methodcomprising: replacing a standard light source with an excitation lightsource for generating a filtered excitation light that maximizes theexcitation of flavoprotein auto-fluorescence and minimizes theexcitation of non-flavoprotein autofluorescence; positioning an imagedetection device to detect a single image representing a retinalauto-fluorescence generated in response to the filtered excitation lightimmediately, to minimize inaccuracies introduced by eye movements andrapid physiological changes; and increasing the intensity of the singleimage using an intensifier.
 28. The method of upgrading a standardimaging device of claim 27, further comprising positioning a filterbetween the image detection device and a subject retina to maximize thepassage of flavoprotein auto-fluorescence and attenuate non-flavoproteinauto-fluorescence.
 29. The method of upgrading a standard imaging deviceof claim 27, wherein providing the excitation light source includesproviding a mercury lamp.
 30. The method of upgrading a standard imagingdevice of claim 27, wherein providing the excitation light sourceincludes providing a laser.
 31. The method of upgrading a standardimaging device of claim 27, wherein generating the filtered excitationlight includes producing light at a wavelength of about 460 nm.
 32. Themethod of upgrading a standard imaging device of claim 27, furthercomprising positioning at least one objective lens to scale the detectedsignal image.